Immunization of chickens with Salmonella enterica subspecies enterica serovar Enteritidis pathogenicity island-2 proteins

Several structural components of the type III secretion systems (T3SS) encoded by Salmonella pathogenicity island (SPI)-1 and SPI-2 are exposed to the host's immune system prior to/during the infection/invasion process, making them potential vaccine candidates. In this study we evaluated whether chickens vaccinated with SPI-2 T3SS components could mount a significant humoral immune response (as measured by serum IgG titres) and whether these antibodies could be transferred to progeny (as measured by egg yolk IgG titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with SE. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgG that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered Salmonella enterica subspecies enterica serovar Enteritidis (SE) per bird in most situations.     

Effects of Salmonella enterica subsp. enterica serovar Enteritidis vaccination in layer hens subjected to S. Enteritidis challenge and various feed withdrawal regimens

Levels of Salmonella enterica subsp. enterica serovar Enteritidis infection and serum S. Enteritidis antibodies after experimental S. Enteritidis challenge and feed withdrawal were investigated in S. Enteritidis-vaccinated and unvaccinated hens. The results were used to determine whether formalin-inactivated S. Enteritidis vaccination can protect layer hens from S. Enteritidis challenge during feed withdrawal periods. S. Enteritidis infection rates were evaluated from cloacal swabs, eggs and organs. Serum antibody titers to deflagellated S. Enteritidis whole cells (DEWC) and S. Enteritidis FliC-specific 9-kDa polypeptide (SEp 9) were examined by commercial ELISA kits. Cloacal S. Enteritidis recovery rates were lower in the vaccinated than unvaccinated group. Recovery rates of S. Enteritidis from samples increased after feed withdrawal and decreased after re-introduction of feed. S. Enteritidis counts in cloacal swabs were lower in the vaccinated than in the unvaccinated group (P<0.05). More S. Enteritidis-positive eggs were detected from the unvaccinated group. Before S. Enteritidis challenge, the DEWC ELISA titer of the vaccinated group was higher (P<0.05) than the unvaccinated group; subsequently, the S. Enteritidis DEWC ELISA titers of both groups increased gradually. In contrast, only the vaccinated group elicited high SEp-9 antibody titer during post-challenge and feed withdrawal. Additionally, vaccinated hens yielded negative S. Enteritidis isolation rates from egg contents. There is a correlation between negative S. Enteritidis isolation rates and high SEp 9 titers in vaccinated layer hens challenged with S. Enteritidis and subjected to feed withdrawal regimens. These findings suggest the S. Enteritidis vaccination of pullets may protect against S. Enteritidis infection during forced molting and that SEp 9 titer could be a potential indicator of antibody protection against S. Enteritidis infection. The potential of the SEp 9 peptide as an antigen for S. Enteritidis vaccination in the future is worth noting.     

Efficacy of soluble recombinant FliC protein from Salmonella enterica serovar enteritidis as a potential vaccine candidate against homologous challenge in chickens

FliC, the flagellin antigen of Salmonella Enteritidis, was tested as a vaccine candidate for protective effect against a homologous challenge in chickens. After immunization with recombinant FliC (rFliC) or administration of phosphate-buffered saline (PBS) at 56 days old, the chickens were challenged with 10(9) colony-forming units of Salmonella Enteritidis at 76 days old. The vaccinated birds showed significantly decreased bacterial counts in the liver and cecal contents compared to those administered PBS at 7 days postchallenge, but the protection was partial. The replication experiment also showed a similar result. In both experiments, vaccination induced an increased level of serum anti-rFliC IgG, which was also reactive to the native flagella. The intestinal IgA level was slightly higher in the vaccinated birds than in the control. However, neither the proliferative response nor interferon-gamma secretion of splenic cells upon stimulation with rFliC was induced. Therefore, the effect of rFliC as a vaccine is limited, and further improvement is needed.     

A laboratory study of an inactivated bivalent iron restricted Salmonella enterica serovars Enteritidis and Typhimurium dual vaccine against Typhimurium challenge in chickens

A commercial inactivated iron restricted Salmonella Typhimurium and Salmonella Enteritidis vaccine was used to vaccinate chicks at 1 day and again at 4 weeks of age, with challenge by a high and a low dose of S. Typhimurium given either orally or by contact with seeder birds inoculated orally with a high dose of S. Typhimurium. In all three challenge regimes, the shedding of challenge strain was reduced significantly (p < 0.05) in vaccinated birds compared with unvaccinated controls. Vaccination reduced colonisation of internal organs after challenge by contact seeder birds. However, no effect of vaccination upon colonisation of internal organs after either high or low oral challenge was apparent. In conclusion, the data indicate that the vaccine should be a useful tool in the control of S. Typhimurium infection in chickens.     

The SEF14 fimbrial antigen of Salmonella enterica serovar Enteritidis is encoded within a pathogenicity islet

The DNA sequence of the chromosomal gene cluster encoding the SEF14 fimbriae of Salmonella enterica serovar Enteritidis was determined. Five contiguous open reading frames, sefABCDE, were identified. The sefE gene shared significant homology with araC-like positive regulators. Serovar-associated virulence plasmid (SAP) genes orf7,8,9 and pefI were identified immediately adjacent to the sef operon. The pefI gene encoded a putative regulator of the Plasmid-encoded fimbrial antigen (PEF) expression. The entire sef--pef region, flanked by two IS-like elements, was inserted adjacent to leuX that encoded a transfer RNA molecule. The organisation of this region was suggestive of a classic pathogenicity islet. Southern hybridisation confirmed two copies of the SAP derived orf7,8,9 and pefI region in S. Enteritidis, one in the chromosome and one on the SAP. Of other group D Salmonella, only S. Blegdam and S. Moscow harboured both chromosomal and plasmid copies of pefI--orf9 region although polymorphism was evident.     

Decreased colonization of chicks by Salmonella enterica serovar Gallinarum expressing mannose-sensitive FimH adhesin from Salmonella enterica serovar Enteritidis

To investigate the role of non-hemagglutinating type 1 fimbriae in the pathogenesis of Salmonella Gallinarum, the isogenic mutant elaborating type 1 fimbriae with mannose-sensitive (MS) variant of the FimH adhesin from Salmonella Enteritidis and the mutant strain with no FimH expression were constructed. Their binding to chicken leukocytes in vitro and invasiveness in 1-day-old chicks were studied. Our results demonstrated that S. Gallinarum type 1 fimbriae with an endogenous variant of the FimH adhesin mediated mannose-resistant (MR) binding to avian leukocytes and did not bind to human epithelial cells. However, after allelic replacement of the FimH, mutated fimbriae with S. Enteritidis variant of the FimH adhesin bound to both cell types in a mannose-dependent manner. In chick model, S. Gallinarum expressing wild-type FimH variant colonized cecal tonsils and bursa of Fabricius more effectively and invaded the spleen and liver in greater numbers than S. Gallinarum fimH knockout strain or mutant expressing MS FimH variant from S. Enteritidis. The invasive potential of the latter was greatly reduced in chicks since no viable bacteria expressing MS variant of the adhesin could be recovered from intestinal lymphoid tissues or liver over a 6 days course of infection. Together, these results demonstrate that the S. Gallinarum type 1 fimbriae with the endogenous MR variant of the FimH protein increase systemic dissemination of S. Gallinarum and colonization of internal organs in chicks indicating the importance of these adhesive structures in the virulence of S. Gallinarum.     

Salmonella in poultry flocks and humans--S. enterica subspecies enterica serovar Enteritidis in the past

After importing of breeder lines for laying flocks from Canada into the former GDR in 1966 the egg industry in this country was completely isolated from that in Western Germany or other Western European countries until opening the border in Germany in 1989. Because of this isolation from other countries, an analysis of the clonal diversity of Salmonella (S.) Enteritidis isolates originated from humans, chickens and food in the former GDR during the 1980s would provide a unique opportunity to obtain new insight into factors that may have triggered the S. Enteritidis epidemic. While isolates had previously been typed by the phage typing scheme of Lalko and Laszlo we applied for the first time the extended phage typing scheme by Ward for the retrospective analysis of the S. Enteritidis strains. Furthermore, isolates of phage type (PT) 4/6 (Ward / Lalko and Laszlo) from different livestocks were investigated by ribotyping. Although in total the PT4/6 dominated between 1986 and 1989 in poultry, other phage types have prevailed in the early 1980s and represented a considerable fraction of isolates until 1989. For instance, PT8/7 was isolated from one large layer farm (district Halle) from 1988 until 1989. During that time in another farm (district Cottbus) only PT8/7 was detected too. PT4/6 was isolated from neither of these two laying hen farms. The strains of PT4/6 could be distinguished by ribotyping in 19 different subtypes. The strains from the northern farms were distinct from those isolated in the southern regions. As farms which were harbouring either PT4/6 or PT8/7 had obtained laying hens from the same sources (breeder lines in Deersheim and Spreenhagen) it is highly probable that S. Enteritidis infection was acquired from the environment at each individual farm. This conclusion is also supported by the presence of different PT4/6 ribotypes in different farms. The presence of different phage types or PT4/6 ribotypes at different farms of laying hens suggests that in each case the S. Enteritidis strains present in the environment were able to enter chicken flocks.     

Immunogenicity of a Salmonella Enteritidis mutant as vaccine candidate and its protective efficacy against salmonellosis in chickens

A novel Salmonella Enteritidis (SE) vaccine candidate strain, JOL919 was constructed by deleting the lon and cpxR genes from a wild-type SE using an allelic exchange method. The study was carried out to evaluate the strain as a vaccine candidate against salmonellosis. The strain showed the enhanced macrophage invasion, early bacterial clearance and higher immune responses as compared to the other mutants, JOL917 (Δlon) and JOL918 (ΔcpxR), and the wild type. In further analysis, the chickens immunized with JOL919 showed a significant increase in plasma IgG and intestinal secretory IgA levels, which was an indication of robust humoral and mucosal immune responses induced by the candidate. The lymphocyte proliferation response and CD45(+)CD3(+) T cells, associated with an activation of T helper and cytotoxic cells, were also significantly increased in the immunized group, which indicated that the candidate also induced cellular immune responses. The immune cell influx into caecal tissues analyzed by immunohistochemistry showed that CD8(+) T cells were predominated in the immunized group, suggesting that the candidate can clear the invaded pathogen in the intestines by a more direct way involving cytotoxic activity. By the examination of the protection efficacy measured by observations of gross lesions in the organs and bacterial recovery, the candidate can provide an efficient protection upon virulent challenge.     

Multi-locus sequence typing of Salmonella enterica subsp. enterica serovar Enteritidis strains in Japan between 1973 and 2004

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was responsible for a worldwide pandemic during the 1980s and 1990s; however, changes in the dominant lineage before and after this event remain unknown. This study determined S. Enteritidis lineages before and after this pandemic event in Japan using multilocus sequence typing (MLST). Thirty S. Enteritidis strains were collected in Japan between 1973 and 2004, consisting of 27 human strains from individual episodes, a bovine strain, a liquid egg strain and an eggshell strain. Strains showed nine phage types and 17 pulsed-field profiles with pulsed-field gel electrophoresis. All strains had homologous type 11 sequences without any nucleotide differences in seven housekeeping genes. These MLST results suggest that S. Enteritidis with the diversities revealed by phage typing and pulsed-field profiling has a highly clonal population. Although type 11 S. Enteritidis may exhibit both pleiotropic surface structure and pulsed-field type variation, it is likely to be a stable lineage derived from an ancestor before the 1980s and/or 1990s pandemic in Japan.     

Comparison of real-time PCR with conventional PCR and culture to assess the efficacy of a live attenuated Salmonella enterica serovar Typhimurium vaccine against Salmonella enterica serovar Enteritidis in commercial leghorn chicks vaccinated under field and laboratory conditions

The efficacy of a live attenuated Salmonella Typhimurium Megan Vac 1 vaccine (MV1) was evaluated against Salmonella Enteritidis in chicken pullets with the use of PCR and culture methods. Two hundred Hyline W-32 white leghorn chicks were obtained from a local hatchery and divided into four treatment groups. Two of the groups served as positive and negative controls. The MV1 vaccine was administered to the chicks in the remaining two groups at 1 and 35 days old by either the coarse spray (field) or the oral route (laboratory) method. The chicks were challenged with a high dose of a Salmonella Enteritidis strain at 10 wk old and euthanatized 3 days postinoculation. Samples for PCR analysis were collected prior to enrichment, after pre-enrichment in buffered peptone water (BPW) and after primary enrichment from the ceca, liver, and spleen. None of the samples tested yielded positive results for the Salmonella Typhimurium vaccine strain by either the culture or PCR methods. Results from the standard culture method showed that vaccinating the birds with MV1 reduced the counts of Salmonella Enteritidis recovered from the challenged birds. In addition, fewer pre-enriched samples tested positive for Salmonella Enteritidis among the challenged groups that were vaccinated when compared to the unvaccinated challenged group. Under the conditions of this study, MV1 was unable to prevent colonization of other internal organs such as the liver and spleen. Real-time PCR was significantly more sensitive than conventional PCR (C-PCR) prior to enrichment, but after enrichment the sensitivities of the two methods were similar. Enrichment significantly increased the sensitivity of both PCR methods for the detection of Salmonella Enteritidis in cecal samples, but did not significantly increase the sensitivity for detection of Salmonella Enteritidis in liver and spleen samples that were pre-enriched in BPW. There was no significant difference between the laboratory or field vaccination methods with respect to either the prevalence of Salmonella Enteritidis isolation or the bacterial loads in culture-positive samples. Collectively, the data suggest that MV1 offered some protection against Salmonella Enteritidis in commercial layer chick pullets under the conditions of this study. Given the labor and time required to perform the C-PCR and culture methods, the real-time PCR method may prove to be a more useful method to use in diagnostics.     

Flow cytometric characterization of Peyer's patch and cecal tonsil T lymphocytes in laying hens following challenge with Salmonella enterica serovar Enteritidis

Two trials were conducted to determine T cell changes in Peyer's patches (PP) and cecal tonsils (CT) of specific-pathogen-free Single-Comb White Leghorn hens challenged with Salmonella enterica serovar Enteritidis (SE). Each week, crop lavage samples were obtained from 4 or 3 hens in Trials 1 and 2, respectively. These birds were then sacrificed and their intestinal tracts excised. The crop sample and contents of one cecum from each hen were cultured for the presence of SE. Cells were purified from proximal and distal PP along with both CT and then aliquots of cells were incubated with antibodies to CD4, CD8, and the three T cell receptors (TCR). The T subsets were identified via flow cytometric analysis. Crop and cecal samples were 100% culture positive for SE at week 1 post challenge and a percentage of samples remained positive throughout the study. Some differences in TCR subsets between or within tissues were observed at various times relative to SE challenge but over-all the subsets remained similar during the study. The predominant TCR was TCR2 (vβ1) followed by TCR3 (vβ2). Low numbers of TCR1 (γδ) cells were observed. CD4/CD8 ratios increased in the PP and CT tissues by week 1 post challenge and the ratio elevation persisted throughout the experiment. These results indicate that T cell populations are comparable between PP and CT and enteric SE infection can affect the cellular dynamics of these lymphoid tissues.     

Comparison of methods for differentiation of Salmonella enterica serovar Enteritidis phage type 4 isolates

OBJECTIVE: To compare molecular typing methods for the differentiation of Salmonella enterica serovar Enteritidis phage type (PT) 4 isolates that allowed for the determination of their genetic relatedness. SAMPLE POPULATION: 27 Salmonella Enteritidis PT 4 strains isolated in the United States and Europe. PROCEDURE: Several molecular typing methods were performed to assess their ability to genetically differentiate among Salmonella Enteritidis PT 4 isolates. Results of pulse-field gel electrophoresis (PFGE), repetitive polymerase chain reaction (PCR) assay, 16S rRNA gene sequencing, random amplification of polymorphic DNA (RAPD), PCR-restriction fragment length polymorphism of 16S rRNA, and antimicrobial susceptibility were evaluated. RESULTS: Compared with results for other techniques, results for the RAPD typing method with the RAPD1 primer reveal that it was the most discriminatory fingerprinting technique, and it allowed us to cluster Salmonella Enteritidis PT 4 isolates on the basis of their genetic similarity. CONCLUSIONS AND CLINICAL RELEVANCE: This study revealed the value of RAPD with the RAPD1 primer as a tool for epidemiologic investigations of Salmonella Enteritidis PT 4. It can be used in conjunction with PFGE and phage typing to determine the genetic relatedness of Salmonella Enteritidis isolates involved in outbreaks of disease. A reliable and highly discriminatory method for epidemiologic investigations is critical to allow investigators to identify the source of infections and consequently prevent the spread of Salmonella Enteritidis PT 4.     

Salmonella enterica serovar Enteritidis colonization of the chicken caecum requires the HilA regulatory protein

Invasion of Salmonella into intestinal epithelial cells is believed to be essential for the pathogenesis of Salmonella infections. Invasion is mediated by genes located on the Salmonella pathogenicity Island I (SPI-1), which are needed for assembling a type three secretion system, that mediates injection of bacterial proteins into the cytosol of epithelial cells, resulting in cytoskeletal rearrangements and as a consequence invasion. HilA is the key regulator of the Salmonella Pathogenicity Island I. To assess the role of hilA in colonization of gut and internal organs in poultry, animals were infected with 10(8) CFU of a delta hilA mutant of S. Enteritidis and its parent strain at day of hatch. Very low numbers of delta hilA mutant strain were able to colonize the internal organs shortly after infection, but they were not eliminated from internal organs at 4 weeks post-infection. At that time, the colonization level of the wild type bacteria in internal organs was decreased to the same low level compared with delta hilA mutant strain bacteria. Shedding of the delta hilA mutant strain and colonization of the caeca was seriously decreased relative to the parent strain starting from Day 5 post-infection. At 4 weeks post-infection, the delta hilA mutant strain was more or less eliminated from the chicken gut, while the parent strain was still shed to a high level and colonized the caeca to a high extent (more than 10(7) CFU/g). It is concluded that hilA is involved in long-term shedding and colonization of S. Enteritidis in the chicken caeca.     

Eggshell penetration of hen's eggs by Salmonella enterica serovar Enteritidis upon various storage conditions

1. The survival and penetration of Salmonella enterica serovar Enteritidis (SE) inoculated on the eggshell was examined upon storage for up to 20 d at real-life conditions (15 to 25 degrees C and 45 to 75% relative humidity (RH)). 2. Penetration was assessed by emptying the egg contents and filling the eggs with a selective medium that allowed visualising Salmonella growth on the inside of the shell and membrane complex. 3. The study of survival on the eggshells was based on viable counts and showed that numbers of surviving organisms decreased over time. Survival was inversely related to storage temperature and RH. Although the average counts decreased over time, a limited proportion of shells carried high numbers of SE at all storage conditions. 4. Penetration spots were observed earlier using an increased storage temperature due to increased growth rates of SE on the agar. After 20 d of storage a similar percentage (c. 44.7%) of eggshells became penetrated, irrespective of the storage conditions tested in this study. 5. The higher the Salmonella shell contamination at the end of storage, the higher the probability that the eggshell was penetrated. Salmonella shell counts exceeding 4 log cfu yielded more than a 90% probability of eggshell penetration occurring.     

Salmonella enterica serovar Typhimurium and Enteritidis infection of pigs and cytokine signalling in palatine tonsils

Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1β and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.     

Phage types of Salmonella enterica serovar Enteritidis isolated in South Africa from 1991-1995

A total of 615 strains of Salmonella enterica serovar Enteritidis (SE), received from 1991-1995 at the Onderstepoort Veterinary Institute (OVI), were phage typed. Most SE isolates (54,7%) originated from poultry followed by humans (28,5 %) and poultry eggs (9,6 %). Phage type 34 was the most prevalent (40,5 %) of all isolates, followed by phage type 4 (33,8 %). Other phage types identified were 1, lb, 4a, 7, 7a, 9a, 14, 24, 24var and 35 (in total 2,4% of isolates). Most isolates of SE were received from the Western Cape Province (47,4%) and Gauteng (22,3%). In poultry phage type 4 was dominant, but in humans, eggs, goats, ducks, sheep, pigs and rabbits, phage type 34 was the dominant type. It appeared as if the poultry-associated epidemic of SE in South Africa that occurred from 1991-1995 originated in the Western Cape Province during 1991 amongst poultry and then spread from there to humans and eggs and then to the rest of the country, where it emerged during 1993. Results indicate that phage type 34 was the dominant phage type from 1991-1993, but during 1994-1995 its presence declined. During this latter period the presence of phage type 4 increased. This may suggest that two smaller epidemics consisting of the two different phage types might have been responsible for the epidemic that occurred from 1991-1995.     

Typing of Salmonella enterica subsp. enterica serovar Mbandaka isolates

Recently, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has gained some importance in the epidemiology of salmonellosis in Poland. Since biotyping, resistance typing, and plasmid profiling were insufficient for strain differentiation, genome macrorestriction by means of pulse-field gel electrophoresis (PFGE) was applied and proved to be the method of choice in S. Mbandaka epidemiological studies. XbaI and BcuI macrorestriction produced 15 and 14 pulse-field profiles (PFP), respectively, but in the case of each enzyme one profile was prevalent. When macrorestriction profiles were combined, a total 24 patterns were found. Based on the similarity of the profiles, four clonal lineages were identified. One clonal lineage contained the majority of poultry, feed and human isolates. Poultry was concluded to be an important source of S. Mbandaka for humans in Poland. Complementary use of various typing techniques improved efficacy of epidemiological studies giving possibility to subdivide S. Mbandaka into 35 types and the index of discrimination reached 0.947.     

Efficacy of Eugenol Against a Salmonella enterica serovar Enteritidis Experimental Infection in Commercial Layers in Production

An experimental study (15-wk-old ISA Brown pullets) was conducted to establish the efficacy of the essential oil of Eugenia caryophyllata against Salmonella enterica serovar Enteritidis. The trial was composed of 4 groups. Pullets in groups 3 and 4 were fed with a commercial compound feed, and pullets in groups 1 and 2 were fed with the same feed plus the aromatic product at the dose of 250 g/Tm. At 19 wk old, the pullets in groups 1 and 3 were infected individually with an inoculum of 3.2 ± 0.8 × 10⁷ cfu of Salmonella enterica serovar Enteritidis/ pullet. During the postinoculation period, samples of feces and eggs were cultured, and pullets were killed 30 d postinoculation. The aromatic product containing eugenol seems to aid in the cleaning of the intestinal and systemic infections, and it also plays an important role in the control of Salmonella cross contamination in eggs.